1. Field of the Invention
This invention relates to methods and compositions for isolating, growing and transforming kidney tubule stem cells.
2. Discussion of the Background
Renal failure is a common clinical syndrome, defined as a decline in renal function, either acutely or chronically. The clinical manifestations of this disorder arise from a decline in glomerular filtration rate and an inability of the kidney to excrete the toxic metabolic wastes produced by the body. The complete treatment of this condition is dependent upon the replacement of filtrative, reabsorptive, homeostatic and endocrine functions of the kidney as an integrated organ structure.
In this regard, the function of a tissue is critically dependent upon the spatial arrangement of its constituent cells. The precise molecular determinants of such pattern formation both in vitro and in vivo is complicated, but soluble factors, such as growth factors, and insoluble factors, such as extracellular matrix molecules, most likely play fundamental roles in this process. Soluble molecules include both growth promoters (epidermal growth factor) and growth inhibitors (transforming growth factor-xcex2). Insoluble factors include complex extracellular matrices (collagen gels, Matrigel) or extracellular matrix (ECM) molecules (laminin, fibronectin, collagen types I and IV).
This critical interplay of structure and function is demonstrated in the embryonic morphogenesis of the kidney, which is dependent upon a finely orchestrated interaction between mesenchyme and epithelium. The initial steps in differentiated nephrogenesis are followed by the development of tubule epithelial cell polarity and lumen formation. Coincident with the onset of cell polarity and tubulogenesis, as defined by both morphologic and directional transport properties, is the appearance of the A chain of laminin, a cell attachment protein of the basement membrane, in the basal regions of the mesenchymal cell aggregate. A sequential series of growth and further differentiation processes then follows to result eventually in a fully formed and functional kidney.
If this complex process of kidney organogenesis could be mimicked in vitro, novel methods for the treatment of renal failure could become available. Various tissue engineering products could be constructed from both semi-synthetic and organic components for complete replacement of renal function in patients with renal failure. Such advances might also allow the development of bioreactors comprising purely organic material for the substitution of renal function in a patient whose kidneys are compromised. These developments could also allow for kidney organogenesis, resulting in the growth of an organic kidney in vitro, from the isolation of renal tubule stem cells from a donor and subsequent growth and differentiation. The in vitro kidney could later be transplanted to the donor of the original renal cells, resulting in replacement of renal function without any fear of transplant rejection or immunosuppressive therapy. The availability of renal tubule stem cells could also promote an efficient process for incorporation of various genes into renal cells for gene therapy of various diseases.
However, such developments are predicated upon the development of a culture system which allows for isolation and growth of kidney tubule stem cells and for in vitro tubulogenesis.
Such a culture system has not-been achieved in the prior art, although in vivo kidney cells have demonstrated a potential for differentiation and regeneration. As demonstrated by Humes et al., J. Clin. Invest. 84:1757-61 (1989) and by Coimbra et al., Am. J. Physiol. 259:F438-F443 (1990), complete recovery of renal function can occur after severe nephrotoxic or ischemic acute renal injury that was of a magnitude to produce complete renal failure. Thus, some subset of renal proximal tubule cells apparently has the ability in vivo to regenerate and form a fully functional, differentiated epithelium. However, such results are doubtlessly the result of the complex interaction of a large number of biological factors responsible for growth, differentiation, pattern formation and morphogenesis of the renal tubule.
Certain of these factors have been identified and employed in renal cell cultures. TGF-xcex21 has been recently shown to transform a monolayer of renal proximal tubule cells in primary culture into a three-dimensional adhesive aggregate of cells, see Humes et al, Lab. Invest. 64:538-545 (1991). EGF has been shown to be a potent growth promoter for renal epithelial cells, see Norman et al, Am. J. Physiol. 253:F299-F309 (1987). Retinoic acid has been reported to increase laminin synthesis in embryonic cell lines by promoting laminin gene transcription, see Dziadpk et al, Devel. Biol. 111:372-382 (1985); Vasios et al, Proc. Natl. Acad. Sci. (USA) 86:9099-9103 (1989); and Rogers et al, J. Cell. Biol. 110:1767-1777 (1990). However, these efforts of the prior art have all failed to evoke tubulogenesis in renal cell cultures. Such tubulogenesis is the first step towards in vitro kidney organogenesis.
Accordingly, it is an object of this invention to provide novel methods, including culture media conditions, for in vitro kidney tubulogenesis.
It is another object of the present invention to provide novel methods for isolating and growing kidney tubule stem cells.
It is another object of this invention to provide a novel, functioning ex vivo kidney tubule tissue system.
It is another object of this invention to provide novel methods, including culture media conditions, for culturing renal tubule stem cells so as to effect gene therapy upon said cells.
The present invention provides methods and composition which satisfy all of the above objects of this invention, and other objects, as will be apparent from the description of the invention given hereinbelow.
The present invention is based on the inventor""s discovery of novel methods, including culture media conditions, which provide for isolation and growth of kidney tubule stem cells, for in vitro kidney tubulogenesis and for ex vivo construction of renal tubules. These methods rely on culturing renal proximal tubule cells in a hormonally-defined culture medium which is treated with combinations of transforming growth factor-xcex21 (TGF-xcex21), epidermal growth factor (EGF) and the retinoid, all-trans retinoic acid (RA), while maintaining the culture under physiologically acceptable conditions.
The inventor has discovered that the administration of TGF-xcex21, EGF and all-trans retinoic acid transform a confluent monolayer of renal proximal tubule cells into three-dimensional cell aggregates containing lumens within the interior of the cell clusters. The lumens were bordered by tubule cells possessing a polarized epithelial cell phenotype with extensive microvilli formation and tight junctional complexes along the luminal border.
In one embodiment all three factors are used to isolate and grow kidney tubule stem cells and to induce this phenotypic transformation. In an alternate embodiment all-trans retinoic acid and EGF are employed to isolate and grow kidney tubule stem cells and to induce tubulogenesis. It is possible to substitute transforming growth factor-xcex1 for EGF. These results demonstrate that the growth factors, TGF-xcex21 and EGF, and the retinoid, all-trans retinoic acid, promote tubulogenesis of adult renal proximal tubule cells in kidney cell culture.